Both intrinsic and extrinsic triggers of apoptosis induce a marked apoptosis of iNKT cells in contrast to conventional Vβ2 T cells. (A) Activation-induced cell death of expanded iNKT and Vβ2 T cells from 10 different healthy donors stimulated with anti-CD3 antibody (OKT3). Error bars (SD) correspond to assays done in duplicate. (B) Expression of CD3 on iNKT and Vβ2 cells by flow cytometry. Gray histograms represent the isotype control and the black lines represent the anti-CD3. Data correspond to 1 representative experiment of 10. (C) Same as panel A, except that cells were stimulated with increasing concentrations of anti-CD3 antibody. Data from 1 experiment with 1 healthy donor (No. 6) with assays done in duplicate (error bars correspond to SD). (D) Apoptosis of iNKT cells incubated with RAJI cells expressing CD1d with medium (med) or αGalCer for 24 hours. Data correspond to 1 representative experiment of 3 with 2 different donors (Nos. 9 and 6). (E) Apoptosis of expanded iNKT and Vβ2 T cells from 9 different healthy donors stimulated with soluble anti-FAS antibody (APO1.3). Assays were done in duplicate (error bars correspond to SD). (F) Expression of FAS (CD95) on iNKT and Vβ2 T cells by flow cytometry. Gray histograms represent the isotype control and the black lines represent the anti-CD95 antibody staining. Data correspond to 1 representative experiment of 7. (G) Same as panel E, except that cells from 4 different healthy donors were stimulated with increasing concentrations of APO1.3 in the presence or not of z-VAD. One representative experiment of 4 independent experiments with 4 healthy donors (Nos. 3, 4, 6, and 20) is showed with assays done in duplicate. (H) Etoposide-induced apoptosis of expanded cells from 1 healthy donor (No. 4). With the highest concentration of etoposide, cells were also incubated with z-VAD (right panel). (I) Puromycin-induced apoptosis. Data are mean ± SD of 3 experiments with 3 healthy donors (Nos. 5, 6, and 9) with assays done in duplicate. (J) H₂O₂-induced apoptosis. Data are mean ± SD of 2 experiments with 2 healthy donors (Nos. 6 and 9) with assays done in duplicate. (K) Staurosporine-induced apoptosis. Data are mean ± SD of 2 experiments in duplicate with 2 healthy donors (Nos. 7 and 9). See also supplemental Figure 3.